This invention relates to new and improved compositions for and methods of treating humans and other animals to reduce DNA damage, enhance DNA repair capacity, and stimulate immune cellular function. More particularly, it relates to the administration to human (or other animal) subjects of a combination of carotenoid material, nicotinamide material, and zinc source material (all as hereinafter defined), e.g. as a drug treatment or a daily dietary supplement, and to compositions containing that combination of materials.
The term "carotenoid material" as used herein means carotenoids, such as material selected from the group consisting of alpha carotene, beta carotene, gamma carotene, lycopene and mixtures thereof. The term "nicotinamide material" as used herein means material selected from the group consisting of nicotinamide, niacin, tryptophane (an amino acid precursor to niacin synthesis) and mixtures thereof. The term "zinc source material" as used herein means an appropriate source of zinc for administration to humans and/or other animals, e.g. one or more zinc salts, such as zinc sulfate or other zinc salts like amino acids such as methionine or aspartate, dipeptides, gluconates, halides, nitrates, oxides or acetates.
In a specific aspect, the invention relates to a novel combination of naturally occurring carotenoid material, nicotinamide material and zinc source material as a combined treatment to aid patients in resisting cellular DNA damage such as oxidative damage, enhancing cellular DNA repair capacity and stimulating immune cellular function. In another specific sense, this combination of chemicals can be used as a dietary supplementation, or as a drug treatment, to prevent (or improve an individual's ability to resist) DNA damage, enhance DNA repair and stimulate immune function in diseases where these processes are central to the manifested disease state; e.g. ageing, cancer, cardiovascular disease and autoimmune disorders such as diabetes, rheumatoid arthritis and ulcerative colitis (Cross et al., Ann. Int. Med. 107:526-545, 1987; Harris, C. C., Cancer Res. 51(Suppl): 5023s-5044s, 1991; Olin, K. L. et al., Proc. Soc. Expt. Biol and Med. 203(4):461-466, 1993).
Carotenoids (Lupulescu, A., Int. J. Vit. Nutri. Res. 64(1): 3-14, 1993; Prabhala, R. H. et al., Ann. N. Y. Acad. Sci. 691:262-263, 1993; Chew, B. P. J., Dairy Sci. 76(9):2804-2811, 1993; Santamaria, L. et al., J. Nutri. Sci. Vit. Spec. No:321-326, 1992; Machlin, L. J., Crit. Rev. Food Sci. and Nutri. 35 (1-2):41-50, 1995; Murakoshi, M. et al., Cancer Res. 52:6583-6587, 1992; Okuzumi, J. et al., Oncology 49:492-497, 1992), nicotinamide/niacin (Mandrup-Poulsen, T. et al., Diabetes Metabolism Rev. 9(4):295-309, 1993; Pero, R. W. et al., Biochimie, 77:385-393, 1995; Shockett, P. J., Immunol. 151(12):6962-6976, 1993; Boulikas, T., AntiCancer Res. 12(3):885-898, 1992; Brown, R. R., Adv. Expt. Med. Biol. 294:425-435, 1991; Henkin, Y. et al., Amer. J. Med. 91(3):239-246, 1991; Jacobsen, E. L., J. Am. Col. Nutri. 12(4):412-416, 1993) and zinc (Singh, A. et al., J. Appl. Physiology 76(6):2298-2303, 1994; Walsh, C. T. et al., Environmental Health Perspectives 102(Suppl. 2):5-46, 1994; Mocchegiani, E. et al., Blood 83(3):749-757, 1994; Singh, K. P. et al., Immunopharmacol, Immunotoxicol. 14(4):813-840, 1992; Mei, W. et al., Biol. Trace Element Res. 28(1):11-19, 1991; Chandra, R. K. et al., Clin. Lab. Med. 13(2):455-461, 1993) are each individually well recognized to possess disease preventive and immune stimulatory properties, even though they have never been combined with each other as a combination therapy, where they could prevent or delay human diseases and stimulate immune function (Compendium of Nonprescription Products, Canadian Pharmaceutical Association, 1994; Canadian Drug Identification Code, June 1995; The Extra Pharmacopoeia, Martindale, 30th edition; U.S. Pharmacopoeia Dispensing Information, 15th edition, 1995). Hence, it is not obvious from the scientific literature, or from availability of commercial products, that if one combines these agents above the normal dietary levels of carotenoids =as vit. A, 1467.+-.1213 kcal, nicotinamide=33.1.+-.26.7 mg, and zinc=6.8.+-.8.4 mg (Payette, H., Am. J. Clin. Nutr. 52:927-932, 1990) having different mechanisms of action leading to the control of the same type of diseases, there could be achieved a formulation having potent properties in improving an individual's ability to resist cellular DNA damage, enhance cellular DNA repair and stimulate cellular immune function.
Humans have been selected over hundreds of thousands of years to respond to not one chemical but to a myriad of chemicals coming to us through our environment mainly thorough the diet. One can assume that our physiology is extremely well balanced to handle and process these chemical mixtures to extract as efficiently as possible the necessities of life such as nutritional energy sources and chemicals to maintain cellular homeostasis and reproduction. This has to be accomplished without introducing any toxicological consequences. Hence, it follows there is a reasonable likelihood that when humans see natural medicines above the levels normally found in the diet or environment, there exists a strong interaction between the megadoses of natural medicines, so that one supplement limits the uptake and metabolism of another, in an effort to provide a natural selection model by which humans can be protected from the toxicological consequences of overdosing. For example, the practice of prior art teaches that carotenoids and vitamins E or C are all radical (electrophilic) scavengers and that these natural products can be combined into supplements for additive biological effects. However, recent literature has not confirmed this practice based on scientific assumption because it was shown that these radical scavengers could inhibit each other's uptake and negate the desired induction of biological effects (Inform 6(7):778-783, 1995; Zhang et al., J. Clin. Nutr. 62:1477S-1482S, 1995; Niki et al., Am. J. Clin. Nutr. 62:1322S-1326S, 1995).
There are commercial products that are sold which have megadoses (i.e. above dietary levels) of carotenoids, nicotinamide and zinc offered in combination with each other, and in addition, they are formulated with several other chemically elucidated natural products. Two examples are given below:
______________________________________ Commercial Product I ("Radical Fighters .RTM.," Twin Laboratories, Inc., Ronkonko- ma, NY 11779 U.S.A.) Beta carotene (pro-vitamin A eq.) 12500 IU Vitamin C 750 mg Ascorbyl palmitate 125 mg Vitamin E (d-alpha tocopherol) 250 IU L-cysteine 250 mg L-glutathione (reduced) 12.5 mg Selenium (selenite) 100 mcg Zinc (gluconate) 10 mg Vitamin B-1 75 mg Vitamin B-2 50 mg Nicotinamide 50 mg Niacin 25 mg Vitamin B-5 250 mg Vitamin B-6 62.5 mg Vitamin B-12 150 mcg Folic acid 200 mcg Biotin 75 mcg PABA (para-aminobenzoic acid) 150 mg Inositol 100 mg Choline 100 mg Commercial Product II (Vitamins for Women, Bonne Forme, 4250 Hempstead Tpke, Suite 21, Bethpage, NY 11714 USA) Vitamin A 5000 IU Beta carotene 3 mg Vitamin D-3 400 IU Vitamin E 200 IU Vitamin K 10 mcg Vitamin C 500 mg Vitamin B-1 10 mg Vitamin B-2 10 mg Vitamin B-6 50 mg Niacin 50 mg Folic acid 400 mcg Vitamin B-12 50 mcg Biotin 50 mcg Pantothenic acid 20 mg Calcium 1000 mg Magnesium 300 mg Potassium 40 mg Iron 9 mg Zinc 15 mg Copper 1 mg Manganese 5 mg Iodine 50 mcg Chromium 80 mcg Selenium 50 mcg Molybdenum 10 mcg ______________________________________
However, these commercial products do not establish or make obvious that the specific combination of carotenoids, nicotinamide and zinc is effective at reducing cellular DNA damage induction or enhancing DNA repair and immune function. On the contrary, as demonstrated below, applicant herein has now found that the administration of carotenoids, nicotinamide and zinc in combination with other natural medicines or nutrients such as the Commercial Product I referred to above does not reduce cellular DNA damage induction or enhance DNA repair and immune function as has been assumed but not proven in the prior art. This discovery is also consistent with the prior art (Inform 6(7):778-783, 1995; Zhang et al., J. Clin. Nutr. 62:1477S-1482S, 1995; Nidi et al., Am. J. Clin. Nutr. 62:1322S-1326S, 1995) which has confirmed that the natural products (e.g. medicines or nutrients) having similar modes of biochemical action have been shown to block each other's uptake and absorption, thus resulting in altered biological functions. It follows then although not practiced in the prior art that it cannot be assumed supplementing an a priori combination of natural products above dietary levels will result in additive biological effects of each product administered separately without previously establishing the lack of inhibition of natural products supplemented in combination.
The exact mechanism of action of carotenoids such as beta carotene is not fully understood but it is commonly accepted scientifically that one primary mechanism is to scavenge oxygen derived free radicals produced either as by-products of metabolism or from exogenous environmental exposures (Lieber, D. C., Ann. N. Y. Acad. Sci. 691:20-31, 1993; Bohm, F. et al., J. Photochem. Photobiol. 21(2-3):219-221, 1993; Regnault, C. et al., Ann. Pharmacotherapy 27(11):1349-1350, 1993). As a free radical scavenger, carotenoids can be expected to reduce or protect against the chemical damage induced in DNA, RNA and protein of cells by toxic environmental exposures or endogenous cellular metabolic errors that ultimately can result in a disease state. On the other hand, nicotinamide and zinc salts do not possess this chemical property which results in an improved biological cellular function.
Nicotinamide and its metabolic equivalent nicotinic acid (niacin, vitamin B.sub.3) or even tryptophane which is the synthetic precursor to niacin is the main precursor for the formation and maintenance of the cellular pool of NAD (Bernofsky, Mol. Cell. Biochem. 33:135-143, 1980; Olsson, A. et al., Biochem. Pharmacol. 45:1191-1200, 1993). NAD is essential for cellular ATP production and maintenance of the cell's redox potential, and it is also the substrate for the DNA repair enzyme, poly ADP-ribosyl transferase (ADPRT). Niacin deprivation decreases the NAD pools significantly both in tissue culture cells (Jacobson, E. et al., IN: ADP-Ribosylation Reactions (Poirier, G. G. and Moreau, P., eds.), pp. 153-162, Springer Verlag, New York, N.Y., 1992), and animal systems (Zhang et al., J. Nutri. 123:1349-1355, 1993) as well as humans (Fu et al., J. Nutri. 119:1949-1955, 1989). The depleted cells have an increased sensitivity to DNA damage and the levels of poly(ADP-ribose) production in cultured cells (Jacobson, E. L., as cited, 1992) or in rat liver (Rawling et al., J. Nutri. 124:1597-1603, 1994) were significantly lower after mild nicotinamide deficiency. On the other hand, when niacin was given as a supplement to ordinary nutrition (i.e. above known dietary levels) the NAD pool increased and the cells were less sensitive to oxygen radicals (Weitberg, A. B., Mutational Res. 216:197-201, 1989). Therefore, it is obvious from this review of the prior art that the primary mechanism of action of nicotinamide/niacin differs from carotenoids and zinc in that the cell's potential for energy metabolism is increased by amplifying NAD and ATP pool supplies (i.e. these biochemicals are the energy sources of living organisms) which in turn is useful to cells, tissues and organs to reduce DNA damage, enhance DNA repair (i.e. poly ADP-ribosylation) and stimulate immune function where the relevance to the disease state is apparent (Pero, R. W. et al., Biochimie 77:385-393, 1995).
Zinc differs from the carotenoids and nicotinamide with regard to its mechanism of action in that it influences disease development and immune function by being an essential co-factor in several enzyme functions involving replication, DNA repair and antioxidant defense of cells. Zinc is required for cell replication and DNA polymerase activity (Williams, R. O. et al., J. Cell Biol. 58:594-601, 1973). There are two zinc fingers in the DNA binding domain of the poly adenosine diphosphate ribosyl transferase (ADPRT) gene and other DNA repair proteins (Dawat, P. et al., Microbiol. 141 (Pt 2):411-417, 1995; Matsuda, T. et al., J. Biol. Chem. 270(8):4152-4157, 1995; Chiriccolo, M. et al., Mutation Res. 295(3):105-111, 1993) which contain cysteine residues (i.e. an amino acid), and if these cysteine residues are oxidized at their thiol constituents, they would prevent DNA binding and participation in DNA repair (Mazen et al., Nucleic Acid Res. 17:4689-4698, 1989; de Murcia, G. et al., BioEssays 13(9):455-462, 1989; Pero, R. W. et al., Biochimie 77:385-393, 1995; Althaus, F. et al., Mol. Cell. Biochem. 138(1-2):53-59, 1994). Moreover, superoxide dismutase is an antioxidant enzyme protecting cells from the harmful superoxide anion because this radical is a substrate for the enzymatic reaction that also requires zinc as a cofactor (Brunori, M. and Rotilio, G., Methods in Enzymology 105:22-35, 1984).
In summation, even though carotenoids, nicotinamide/niacin and zinc have been shown to have some enabling utility in cell and animal models as single agents in the prevention of certain diseases and in the stimulation of immune function, there has been a lack of corresponding, consistent data in humans (Bodgen, J. D. et al., Amer. J. Clin. Nutri. 48:655-663, 1988; Walsh, C. T. et al., Environmental Health Perspectives 102(Suppl. 2):5-46, 1994). In addition, it is not possible for one skilled in the art to a priori predict whether agents with different mechanisms of action will be synergistic, additive or inhibitory to the biological response they will elicit when given in combination.